Tuneable microfibrillar collagen structures within dense chitosan hydrogels

Chitosan-type I collagen hydrogels are paradigms of polysaccharide-protein assemblies with applications as biomaterials. However, preparing physical hydrogels combining them at comparable, high concentrations (> 20 mg.mL-1) within interpenetrated networks remains challenging. Here, we report the preparation of two different types of hydrogels that form under ammonia action starting form identical mix of concentrated chitosan and collagen solutions. Under ammonia vapours, mix solutions form composite hydrogels, where collagen fibers exhibiting an unusual, branched morphology occupy a chitosan network porosity. Immersion of the mix in liquid ammonia yielded hybrid networks where collagen microfibrils were associated with chitosan nanoaggregates. Structural variations impacted the mechanical behaviour and biological properties, assessed by 2D cultures of fibroblasts, of these hydrogels. Difference in gelation kinetics between the two biomacromolecules under the two processes appeared as a key factor driving the mixed network structuration. This work discloses a new route to obtain dense hydrogels from binary biopolymer systems and offers additional insights on the underlying gelation process.